Field of the Invention
The present invention relates to an in vitro or ex vivo method for preserving and/or keeping alive mammalian, preferably human skin biopsies, enabling to transport it and, if applicable, to culture it. The present invention also comprises a skin biopsy thus preserved and obtained by such a method and relates to its use as a model especially in a kit for screening or selecting cosmetic or therapeutic compounds.
Description of the Related Art
Skin is composed of the superimposition of epidermis and dermis. The epidermis is a stratified multi-layered and squamous epithelium. It forms a barrier which can withstand the damage of desiccation, as well as mechanical, chemical and microbial aggressions. The main cell type forming the epidermis is the keratinocyte. This tissues also comprises other cell populations such as the melanocytes, the Langerhans cells and the Merkel cells (see appendix). The epidermis is conventionally sub-divided into four distinct strata comprising internal layers towards the most superficial layers: the basal layer (a layer), the spinous layer (4-15 layers), the granular layer (1-3 layers) and the thorny layer (5-10 layers). The epidermis rests on the dermis thanks to a basal membrane formed among other things of collagen molecules. The dermis contains very dense vascular and nervous networks as well as the epidermal appendices, keratinised structures extending the epidermis and including the pilous follicles, the sebaceous glands and the sweat glands. The skin rests on a sub-cutaneous tissue, called hypodermis, mainly composed of fat cells playing a part, among other things in the elasticity of the skin and the thermoregulation of the system.
In vitro or ex vivo skin culture systems have long been developed for the academic or applied research (Lebonvallet et al. The evolution and use of skin explants: potential and limitations for dermatological research.” Eur J Dermatol 2010; 20 (6): 671-84). First of all, the ban of animal use for the development of cosmetic products in Europe (Pauwel M and Rogiers V. Human health safety evaluation of cosmetics in the EU: a legally imposed challenge to science. Toxicol Appl Pharmacol. 2010 Mar. 1; 243(2): 260-74), as well as the growing importance of the 3R-rule (Reducing, Replacing, Diminishing) relating to animal experimentation during pharmaceutical development (Wells D J. Animal welfare and the 3Rs in European biomedical research. Ann N Y Acad Sci. 2011 December; 1245:14-6), confer to in vitro or ex vivo skin culture systems, in particular those enabling human skin culture, a strategic importance for cosmetic, chemical and pharmaceutical industries. In vitro or ex vivo skin culture systems are ideal models for the study of cutaneous biology. Indeed, they possess all the cell types of the skin, organised into a 3D structure. They reflect directly the parameters of the individuals, such as the age, the sex, the pathological state of the skin or sun exposure (Lebonvallet et al., 2010).
If the in vitro or ex vivo culture systems or devices are ideal models for research, the existing systems or devices are ill-suited for transporting them or for keeping them alive.
Several, more or less perfected culture systems or devices exist (Lebonvallet et al., 2010), however these systems or devices are generally fragile and cannot be shipped by road or plane while maintaining their integrity. Indeed, in these systems or devices, the skin biopsy is not held firmly by a physical support. It can be left to float in a cultivation medium solution or laid on an insert with a porous membrane or a stainless steel grid. The advantage of the last two methods is that the superficial part of the epidermis, the thorny layer, is kept directly in contact with the atmospheric air. This enables to study the topical application of substance at the surface of the skin.
We may also mention here the patent documents published under the numbers EP 2 019 316, US2011/045477, or WO2004/092354 which describe in vitro or ex vivo skin culture systems, but which do not contain any support or device for maintaining or transporting skin biopsies.
More recently, a stainless steel chamber system has been developed for holding firmly large skin biopsies and enable the in vitro or ex vivo culture up to four weeks (Lars Steinstraesser et al. A Human Full-Skin Culture System for Interventional Studies.” Eplasty. 2009; 9: e5.). It may well be interesting enough, but this system is hardly suited for cell due to its dimensions and its composition (stainless steel). Moreover, this system is based on significant tensioning of the skin which does not reflect the physiological conditions encountered for this in vivo organ. EP 0 702 081 may also be quoted which describes a skin model comprising two types of collagen sponges of different density, Inoculated with fibroblasts and keratinocytes. This model can be placed in the centre of a well and surrounded with clotable gelatin. The publication of Jacobs et al. (Methyl green-pyronine staining of porcins organotypic skin explant cultures: an alternative model for screening for skin irritants, ATLA, 28,279-92, 2000) describes the use of pig skin biopsies placed in a culture medium, treated with an irritant, then included in a solid matrix, before being frozen and coloured. US 2003/040113 describes a multi-layered support for cell culture, in which the cell support is seeded with living cells suspended in a gel. WO 2004/022696 describes a fibrin support, comprising fibrinogen and thrombin, used for cell culture, especially keratinocytes. Finally, WO 2012/059703, filed before the present application and published on 10 May 2012, i.e. after filing the present application, describes a method for preserving and/or keeping alive the epidermis.
These documents do not divulge nor suggest a method for keeping alive and transporting a skin fragment or biopsy laid in a liquid matrix which is capable of solidifying, and hence held firmly by the matrix but also nourished, whereas the assembly is contained in an insert whose bottom is formed of a porous membrane.
As regards skin biopsies, the study of certain culture media used for keeping alive in vitro or ex vivo these skin biopsies have emphasised the deleterious effect of the presence of serum on the structure of the skin, whereas the presence of calcium at a concentration of 1.4 mM is important for the cohesion of the tissue via the stimulation of the production of extracellular matrix and of the epidermal differentiation (Lebonvallet et al., 2010).
For many years, we have sought to develop new methods so as to be able to preserve and/or keep alive mammalian, preferably human or pig, skin biopsies. All the more so since these methods enable transport and culture of these biopsies.
The development of such models is indeed quite important for dermatological research and for the studies necessary to the preparation of pharmaceutical and/or cosmetic products.
Thus, it would be desirable to have such a system, device and/or method available to obtain such skin biopsies, which are sufficiently heavy-duty to transport said biopsies (by air and/or by land).
Obtaining such a model would enable to preserve the 3D-organisation and the skin functions from a biopsy, and this advantageously with respect to the cultures of reconstructed skin, which normally take a long time to be developed and require complex techniques (whereas biopsies are easier to obtain and with a larger potential in terms of dermatological research, in particular for the studies on the extracellular matrix, 3D-structure or the interactions between the different types of cutaneous cells).
Such models would advantageously enable the realisation of studies necessary to better understanding of the role of the skin, especially of its epidermis and of the dermis, in the mechanical field as well as in the physiological field. Such biopsies, held firmly thanks to such as heavy-duty system, device and/or method, would also enable to sample cell layers from the thorny layer of the epidermis by successive superficial separations by tape stripping. Such biopsies, thus preserved and transportable, can also be used as ideal models, compared to the models of reconstructed skin and their shortcomings (see Lebonvallet et al., 2010) for predicting by in vitro or ex vivo tests, of the activity of cosmetic and/or pharmaceutical active principles or still of the secondary effects of topical compounds.